Cyclosporine is a cyclic undecapeptide of fungal origin [U.S. Pat. No. 4,117,118; Ruegger, et al., Helvetica Chimica Acta, Vol. 59 (4), pages 1075-1092 (1976); U.S. Pat. No. 4,289,851; and Traber, et al., Helvetica Chemica Acta, Vol. 60 (4), pages 1247-1255(1977) and Vol. 65 (5), pages 1655-1677(1982)] which is commonly employed as a potent immunosuppressive agent to prevent the rejection of transplanted organs such as kidney, heart, bone marrow, and liver in humans. The effectiveness of cyclosporine has also been investigated in the treatment of conditions such as psoriasis, conjunctivitis, arthritis, nephritis and autoimmune diseases [Donnelly, et al., Therapeutic Drug Monitoring, Vol. 11 (6), pages 696-700(1989)]. While a certain level of cyclosporine must be maintained in the bloodstream to prevent rejection of transplanted organs, nephrotoxicity, hepatotoxicity, and other side effects can result from higher blood levels of the drug or from prolonged exposure. Moreover, distribution and metabolism of cyclosporine varies greatly between individuals as well as in a single individual during the course of therapy. Accordingly, it is necessary to monitor the concentration or level of cyclosporine in biological fluids such as whole blood, plasma, and serum, for proper patient management [Burchart, et al., Drug Intelligence and Clinical Pharmacy, Vol. 20, pages 649-652(1986) and Shaw, et al., Clinical Chemistry, Vol. 33 (7), pages 1269-1288(1987)]. Measurement of cyclosporine in blood, plasma and serum has been complicated, however, by the presence of metabolites of cyclosporine therein [Maurer, et al., Drug Metabolism and Disposition, Vol. 12 ( 10), pages 120-126(1984)], and the toxicities, immunosuppressive activities, and synergistic effects of these metabolites are being investigated [Dindzans, et al., Transplantation Proceedings, Vo. 19 (4), pages 3490-3493 (1987); Yee, et. al., Transplant. Proc., Volume 18, pages 774-776(1986); and Ryffel, et. al., Transplant. Proc., Volume 20 (supplement 2), pages 575-584 (1988)]. Although the measurement of cyclosporine independently from its metabolites is desirable, there is also the need for assays that measure the metabolites as well as the parent drug (Donnelly, et al., supra). The metabolites of cyclosporine that have been identified in which the ring is still intact result from the hydroxylations and demethylations of the parent compound [Maurer, et al., Drug Metabolism and Disposition, 12 (1), pages 120-126 (1984)]. The structures of cyclosporine and some of its major metabolites are of the formula:
__________________________________________________________________________ ##STR1## METABOLITE R.sub.1 R.sub.2 R.sub.3 __________________________________________________________________________ Cyclosporine H CH.sub.3 H AM9 (M-1) H CH.sub.3 OH AM19 (M-8) OH CH.sub.3 OH AM1 (M-17) OH CH.sub.3 H AM1c (M-18) * CH.sub.3 H AM4N (M-21) H H H __________________________________________________________________________ ##STR2##
The structure of cyclosporine may be alternately represented by the formula: ##STR3## wherein "MeBmt" respresents a residue of N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)-threonine; "MeVal" represents a residue of (N)-methyl-(L)-valine; "MeLeu" represents a residue of (N)-methyl-L-leucine; "D-Ala" represents a residue of D-alanine; "Ala" represents a residue of L-alanine; "Val" represents a residue of L-valine; "Abu" represents a residue of L-(alpha)-aminobutyric acid; and "Sar" represents a residue of sarcosine, also known as N-methylglycine. The term "residue" refers to the condensed form of the amino acid found in peptides, and the configuration of the alpha-amino acid is assumed to be L unless a D-configuration is specified. Conventional nomenclature for analogs of cyclosporine are defined herein by reference to the structure of cyclosporine (i.e., cyclosporin A) by first indicating those residues in the molecule which differ from those present in cyclosporine, and then applying the term "cyclosporine" to characterize the remaining residues which are identical to those present in cyclosporine. Thus, [Thr].sup.2 cyclosporine designates the cyclosporine in which the amino acid residue in the 2 position is threonine, i.e., cyclosporin C.
Cyclosporine levels in whole blood, plasma and serum have been measured by high performance liquid chromatogaphy (HPLC) [Lensmeyer, et al., Clinical Chemistry, Vol. 31(2), pages 196-201 (1985)], radioimmunoassay (RIA) utilizing .sup.3 H [Donatsch et al., Journal of Immunoassay, Vol. 2(1), pages 19-32 (1981)] or .sup.125 I [U.S. Pat. No. 4,727,035and Mahoney, et al., Clinical Chemistry, Vol. 31(3), pages 459-462 (1985)], fluorescent immunoassays (U.S. Pat. No. 4,727,035), and by fluorescence polarization immunoassay (FPIA) [Marty, et al., Analytical Letters, Vol. 22(13 & 14), pages 2717-2736 (1989) and European Patent Application Publication No. 283,801]. While the metabolites of cyclosporine can be distinguished from cyclosporine itself according to such HPLC methods, HPLC is nevertheless time and labor intensive, requiring extensive sample preparation and at least thirty minutes to perform the assay. Similarly, RIA assays suffer from the disadvantages of using radioactive materials which require special storage, handling and disposal, and typically require a minimum of two hours to perform.
While fluorescent polarization immunoassays are superior to the methods described above, particularly in ease of use, commercially available polyclonal antibody immunoassays display a lack of specificity for cyclosporine over its metabolites. In this regard, the specificity of immunoassays is dependent upon the antibody used, and the relative affinities of the antibody for cyclosporine, metabolites of cyclosporine, and the labeled form of cyclosporine. Recently, monoclonal antibodies specific for cyclosporine over its metabolites have been described [Quesniaux, et al., Immunology Letters, Vol. 12(1), pages 120-126 (1985), Clinical Chemistry, Vol. 33(1), pages 32-37 (1987), and Molecular Immunology, Vol. 24(11), pages 1159-1168 (1987)], and RIA assays using these antibodies have been found to correlate well with HPLC.
The present invention overcomes the disadvantages of the HPLC and RIA methods described above by providing reagents which are particularly useful in immunoassays, especially fluorescent polarization immunoassays, for detecting cyclosporine or cyclosporine and metabolites of cyclosporine. Moreover, the present invention is an advance over immunoassays for cyclosporine which have been previously described by providing novel tracer compounds for use in immunoassays, particularly fluorescent polarization immunoassays, employing either specific or nonspecific antibodies to detect cyclosporine or cyclosporine and metabolites of cyclosporine in a test sample.